Abstract

Human immunodeficiency virus type 1 (HIV-1) establishes a persistent, nonproductive state within a small population of memory CD4(+) cells. The transcription factor LSF binds to sequences within the HIV-1 long terminal repeat (LTR) initiation region and recruits a second factor, YY1, to the LTR. These factors then cooperatively recruit histone deacetylase 1 to the LTR, resulting in inhibition of transcription. This appears to be one mechanism contributing to HIV persistence within resting CD4(+) T cells. We sought to further detail LSF binding to the HIV-1 LTR and factors that regulate LSF occupancy. We find that LSF binds the LTR as a tetramer and that binding is regulated by phosphorylation mediated by mitogen-activated protein kinases (MAPKs). In vitro, phosphorylation of LSF by Erk decreases binding to the LTR, while binding is increased by p38 phosphorylation. LSF occupancy at LTR chromatin is increased by the p38 agonist anisomycin and decreased by specific p38 inhibition. p38 inhibition also results in increased acetylation of histone H4 at the LTR nucleosome adjacent to the LSF binding site. p38 inhibition also blocked the ability of YY1 to inhibit activation of the integrated HIV promoter. Finally, HIV was recovered from the resting CD4(+) T cells of aviremic, HIV-infected donors upon treatment of these cells with specific inhibitor of p38. These data suggest that the MAPK pathway regulates LSF binding to the LTR and thereby one aspect of the regulation of HIV expression. This mechanism could be exploited as a novel therapeutic target to disrupt latent HIV infection.

Binding of LSF to HIV promoter. (A) A schematic representation of the HIV LTR showing the RCS wild-type and mutant oligonucleotides. The sequence of RCS (gray) shown here extends from −10 to +27 in the HIV LTR and contains a site of transcription initiation (shown by *). The sequence important for LSF binding is in bold and underlined; the three sequences “CNRG” forming part of the LSF binding site are shown in the box. (For the sequence of mutants, see Materials and Methods). (B) EMSA with 90 ng of His-LSF (lanes 2 to 9) were performed using the following probes: RCS wild type (lanes 1 and 2) and its mutants (lanes 3 to 9). Binding is decreased when any one of the three motifs is mutated (M1, M2, or M3), nearly ablated when site 3 and another site is mutated (M2+3 or M1+3), and absent when all three motifs are mutated (M1+2+3). M1+2, motifs 1 and 2 are mutated.

LSF binds as a tetramer to the RCS. (A) Bacterially expressed His-LSF and MBP-LSF were incubated alone or combined in the ratios indicated over the lanes. The mixture was analyzed by EMSA in the presence of RCS oligonucleotide (see in the text the definition of gsu). (B) An enlarged image of the complexes in lanes 4 and 5 shows the formation of five bands, indicated by arrows. Each band corresponds to one of the predicted heteromultimeric or homomultimeric complexes modeled. M in the figure refers to MBP-LSF, and H refers to His-LSF.

LSF is phosphorylated by MAP kinases. ERK and p38 phosphorylate LSF at distinct sites. Activated GST-ERK, GST-JNK, or GST-p38 was incubated with [γ32-P]ATP and purified His-LSF as a substrate, and the reaction products were separated by SDS-PAGE. Radiolabeled LSF was isolated and treated with chymotrypsin for analysis by two-dimensional phosphopeptide mapping. All peptide samples were simultaneously analyzed on the same apparatus. The origins are indicated by dashed circles. Peptides phosphorylated by ERK at S289 and S291 are indicated by spots B and C. Spot A represents a partial digestion product. Peptides phosphorylated by p38 are indicated by spots α, β, and γ. Spot X is an unidentified peptide seen in various intensities in different digests.

LSF binding to the RCS is regulated by MAP kinases. His-LSF (1.5 μg) was in vitro phosphorylated by 1 U of Erk, Jnk, or p38 in a final volume of 50 μl. Two microliters of reaction was withdrawn at specified time intervals and used in a binding reaction with 280-LSF probe (A) or RCS probe (B, C, D, E). The concentration of phosphorylated His-LSF in each lane was 96 ng. (A) EMSA of LSF before (lane 1) and after 10 min of in vitro phosphorylation by the three MAP Kinases. The same result was observed when LSF was phosphorylated for 1 or 5 min or less LSF was added to EMSA (data not shown). (B, C, D) LSF binding to the RCS after phosphorylation by Erk, p38, or Jnk, respectively. His-LSF was incubated in a reaction mixture containing phosphorylation buffer for 10 min without enzymes at 4°C (lane 1) or 30°C (lane 2) before EMSA. The phosphorylation took place at 30°C for 1, 5, and 10 min (lanes 3, 4 and 5, respectively). (E) In vitro phosphorylation of His-LSF by both Erk and p38. The two enzymes were added separately or at the same time to the reaction mixture, which took place for 10 min. The position of the LSF/DNA complex is indicated at the left side of the panel. Unbound RCS oligonucleotide probe is also indicated.

In vitro phosphorylation of MBP-LSF and His-LSF291. (A) One microgram of each of the purified proteins was phosphorylated with 50 units of Erk or p38 in the presence of [γ-32P]ATP at 30°C for 10 min. The phosphorylated product was run in 10% SDS-PAGE. (B) In vitro phosphorylation of MBP-LSF and His-LSF291 by Erk and p38 for 10 min at 30°C followed by EMSA with [γ-32P]ATP RCS probe for 15 min at 4°C.

Inhibitor of p38 and Erk regulate LSF binding to the RCS but not to the canonical LSF-280 binding site found in the SV40 promoter. The nuclear extract was prepared from HeLa-CD4-LTR-CAT cells treated with or without 10 μM of Erk inhibitor, U0126, or p38 inhibitor, SB203580, for 2 h. One hundred thirty nanograms of nuclear extract was loaded per lane in the presence of 4 to 8 fmol of RCS (A) or LSF-280 probes (B). An asterisk indicates the band due to nonspecific binding to RCS seen in nuclear extracts (, ) that increases at longer EMSA incubation times (data not shown).

MAP kinase pathway involving p38 regulates LSF binding to DNA in vivo. HeLa-CD4-LTR-CAT cells were treated with 10 μM of SB203580 or anisomycin for 2 h, after which the ChIP assays were carried out using anti-LSF (A, B) or anti-AcH4 antibodies (C). To compare the relative amounts of chromatin used in the immunoprecipitation, 1% of the input from each sample was amplified by PCR. The figure is representative of three experiments. The p38 inhibitor SB203580 decreased LSF occupancy (A) and increased H4 acetylation at the LTR (C). An agonist of p38, anisomycin, increases LSF occupancy at the LTR (B).

p38 modulates integrated HIV-LTR promoter activity in vivo. HeLa-CD4-LTR-CAT cells were cotransfected with CMV-YY1 and/or CMV-Tat plasmids. The total DNA amount used per transfection was 5 μg. The transfected cells were treated with 10 mM SB203580 for 30 min, and a CAT assay was performed on the cells (see Materials and Methods) using the CAT assay kit. Lane 1, cells transfected with CMV vector alone. Lane 2, cells transfected with CMV-Tat. Lane 3, cells cotransfected with CMV-Tat and CMV-YY1. Lane 4, cells transfected with CMV-Tat and CMV-YY1 and treated with 10 mM SB203580. Lane 5, cells transfected with CMV-Tat and treated with SB203580. Lane 6, cells transfected with empty vector and treated with SB203580. The mean percent acetylation from five different experiments is shown above each lane.

SB239063 does not upregulate cell surface markers of activation or proliferation or surface levels of coreceptors on CD3+ CD4+ cells. Cells were incubated with IL-2 plus either PHA or SB239063 for 72 h as described in Methods and then phenotyped. The results are representative of the analysis of cells from three HIV-seropositive donors.

SB239063 does not enhance de novo HIV infection. Viral outgrowth was measured by p24gag antigen following infection of activated peripheral blood mononuclear cells with HIV-LAI. Means and standard deviations of three independent infections are displayed.